Recent evidence has revealed a novel function for the B2 protein encoded by the nodavirus Flock House Virus (FHV). Previous biochemical, structural and functional data identified B2 as a potent suppressor of the innate immune response to dsRNA (RNA silencing). We have obtained results suggesting that B2 is also required for efficient translation of viral RNAs. The research plan proposed in this application utilizes biochemical, functional and imaging techniques to investigate the role of B2 in viral RNA translation. Aim1. Quantify effect of B2 on cellular protein synthesis and association of FHV RNAs with ribosomes: The ribosomal association of FHV RNA2 and RNAS will be analyzed by quantitative polyribosome analysis in the presence and absence of B2. The impact of B2 RNA-binding affinity on the ribosomal association of these RNA will also be investigated. Aim 2. Determine the mechanism by which B2 modulates viral RNA translation: Two models for B2 function will investigated. In the first model B2 binding to the viral mRNAs protects them from siRNA- induced cleavage. The second model proposes that B2 acts as a direct enhancer of viral translation. Aim 3. Functionally characterize interactions of B2 with RNA: Despite the biochemical, structural and functional data implicating functional importance of B2-RNA binding in B2 function, there is currently no evidence that B2 binds dsRNA in vivo. We will biochemically identify the in vivo dsRNA substrates of B2, and use immunofluorescent microscopy to visualize co-localization of B2 and the RNA substrates in vivo. Additionally we will determine the binding affinity of a set of RNA binding mutants of B2. Recently, we discovered that, in addition to its role as a suppressor of RNA silencing, B2 appears to be involved in viral RNA translation. While it is not surprising that B2 might be a multi-functional protein, it is very interesting that B2 is potentially involved in translation as well as replication. The work presented in this proposal will focus on developing a mechanistic understanding of the role of B2 during the FHV infection cycle. This work has important implications not only regarding how the viral capped, non-adenylated RNAs are translated, but also potentially, the mechanism regulating template usage of the viral RNAs. Since the viral RNAs of all positive strand RNA viruses serve as message for translation of viral proteins, templates for replication, and substrates for virion assembly, productive infection is critically dependent upon tight regulation of the functional states of the viral RNAs. Insights into the mechanisms of this regulation could lead to the development of new antiviral targets. [unreadable] [unreadable] [unreadable]